This invention relates to laser capture microdissection (LCM). More specifically, this disclosure sets forth an improved film for use with laser capture microdissection in which the film is used in contact with the specimen and a coating is provided over the film which prevents non specific transfer of specimen without inhibiting desired laser capture microdissection.
Laser capture microdissection (LCM) is known. Specifically, a sample such as a tissue specimen has multiple cell areas scattered throughout. Taking the case of a biopsy for cancer, these cell areas can include cancer cells, pre-cancer cells, irritated cells, normal cells and other tissue. The diagnostician usually desires only one type of cell or cell portion from one cell areaxe2x80x94say for purposes of this examplexe2x80x94the xe2x80x9cpurexe2x80x9d cancer cells. Further, the diagnostician requires sufficient quantity of the cells from the selected cell areas to perform further meaningful diagnosis. As a consequence, multiple samples of similar cells from one or more specimens are frequently required.
Laser capture microdissection is preferably performed with a transparent low temperature melting film such as (polyethylene/vinyl acetate), CAS 24937-78-8) (also known as EVA) manufactured by the Du Pont Corporation of Wilmington, Del. The reader will understand that many other manufacturers vend similar materials.
Specifically, a specimenxe2x80x94usually on a glass slide is overlaid with the low temperature melting film such as EVA. Thereafter, a cell area of the sample wanted for dissection is visualized and located, preferably through a microscope. When location has occurred, precision heating and melting of the low temperature film occurs overlying the visualized cell area. The precision heating of the low melting point film occurs by having a laser heat that portion of the low melting point film that overlies the cell area targeted for microdissection. At the precision heated area, flow of the melted EVA occurs from the film to the sample onto the visualized cell area. The sample is adhered to the melted EVA upon solidification of the precision melted portion of the film. Thereafter, the film is removed from the sample, preferably by placing the film overlying the specimen under tension and lifting the film away from the sample. Typically, the sample at the adhered identified cell area sticks to the film with the result that a microdissection occurs. The reader will understand that the film a coating can either be stretched or can reside on a support surface.
In LCM, at least a major part of the mechanism of adhesion is mechanical. The heated film overlying the selected cell area flows into and around the specimen portions to be microdissected. Thereafter, the heated and flowed low temperature melt film solidifies. When the film is withdrawn, the physical interference between the flowed and solidified film material and the cell area of the specimen intended for microdissection causes the film when it is withdrawn to xe2x80x9cpullxe2x80x9d the selected cell area from the remainder of the specimen. Microdissection occurs.
The primary reason for low temperature in the melting of the film is to avoid damage to or change the nature of the specimen. EVA, among plastics has a uniquely low melting range, which can be controlled by the manufacture. Such manufacture control can include the addition of a variety of ingredients (e.g. rubber) to adjust the melting point and other properties. The ethylene part of the polymer can be used for property variations.
Understanding this mechanical adhesion, the reader can quickly understand the reason for using a low temperature melt film. It is obviously desired to remove the targeted portion of the specimen for further diagnosis. Where the melted film flows in and around the targeted portion of the specimen, undue heating changes the nature of the targeted cellular material and makes may invalidate subsequent analysis or diagnosis. This subsequent analysis, or diagnosis, includes potentially a variety of methods for research and clinical evaluation, such as genetic, immunological, enzymatic, and protein analysis.
At the same time, precise and precision transfer of the intended cell area is required. Such precise transfer must gather only the identified cell areaxe2x80x94say for the sake of the example discussed abovexe2x80x94the xe2x80x9cpurexe2x80x9d cancer cells. In the LCM, recovery of materials from cell areas other than cellular material within the identified cell area is referred to as xe2x80x9cnon-specific transfer.xe2x80x9d Non specific transfer can be detrimental to further analysis including biological amplification techniques such as PCR. Some applications of LCM are sensitive to very low levels of undesired sample areas.
To avoid non-specific transfer, LCM as currently practiced has divided itself into two broad techniques. In one technique, known as non-contact LCM, the film to which attachment occurs is held spaced a small but constant interval from the sample. When local heating of the EVA or other film utilized occurs, the film expands across the spatial interval, and adheres to the specimen at the visualized portion. When the film is removed, microdissection occurs. A solution to this problem has been to devise various means of spacing the EVA film away from the tissue, so that the EVA contacts the tissue only in the selected spots by its expansion during laser melting.
The EVA expands in a column or pedestal at the area of activation by about 10% to 15%, melts into the tissue sample, and then withdraws away slightly, retaining a microscopic tissue sample upon cooling. The desired spacing of the EVA surface away from the tissue sample is of the order of 10 micrometers. There are various ways of providing the spacing, including putting a spacer film on the EVA, located so the spacer film is in contact with an area of the tissue away from the desired sampling point and holding the EVA surface away from the tissue. With regard to such spacing techniques, non specific transfer may also occur even though a spatial interval is present, due to loose or weakly adhering substances and unevenness of tissue.
It is desirable to press the surface of the EVA against the tissue sample with controlled force, as one of the control parameters of tissue sampling, with the spacing preventing actual contact of the EVA and tissue. The coating of this disclosure may be desirable to prevent non specific transfer in this method also.
It is to be understood that non-contact microdissection is not without problems. Specifically, maintaining the film at a precise closely spaced interval from the specimen at the visualized cell area is difficult. Precision control of the parameters of contact of the low temperature melt film to the sample is difficult.
In another technique of LCM, which is directly applicable to this invention, the film is brought into direct contact with the specimen before melting occurs. In the past, this direct contact with the specimen has caused non-specific transfer. Specifically, the film usedxe2x80x94usually EVAxe2x80x94is naturally tacky. This natural tackiness results from the softening point of the film that is required to minimize damage to the microdissected cell area being removed.
Complicating LCM, the biological specimen is also non-homogenous. The specimen typically contains proteins, carbohydrates, fats oils and other cellular materials in an irregular matrix. Portions of this irregular matrix can preferentially adhere to tacky surfaces of the film without the laser heating. Thus, when the targeted material is adhered in the LCM process and withdrawn from the specimen, undesired adjacent cell areas of the specimen are removed and transported by the film. It is this non-specific transfer which it is the purpose of this invention to avoid.
A low temperature melt film such as EVA is prepared for laser capture microdissection by having a thin specimen non-adhering coating which is hard and non tacky. The hard and non-tacky coating may be in the range of 0.1% to 10% of the total film thickness placed on the sample exposed side of the film. Alternately, a coating may be added to EVA in the range of two to two hundred micro inches. The coating may be applied by any acceptable method including solvent based coatings, laminations and the like. When the coated film is brought into contact with the specimen, the specimen non-adhering coating prevents non-specific transfer due to sticky adherence of portions of the sample. Further, the hard coating will allow use of techniques to remove non-specific transferred material, such as by brushing or washing away attracted material from the hard coating. At the same time, the non-specimen adhering coating on the low temperature melt film surface can stabilize and protect the low temperature melt film against variations in performance due to ambient humidity and temperature variation. Upon appropriate heating for laser capture microdissection, the barrier of the thin coating allows conventional film melting with otherwise uninhibited adhesion of selected cell areas to the film. Coatings on the low temperature melt film (EVA) surfaces in selected locations are made, for example by applying film-forming material from a volatile solvent-based solution, followed by evaporation of the solvent. The coating solution can be applied by spraying, dipping, or adding exact volumes to a surface with a micropipet. Spreading a measured volume of solution over the surfaces can coat surfaces. The volume can control the coating thickness and concentration of coating solids added to a known area.
One of the simplest and most practical ways of making a coating on the EVA surface in selected locations is by applying an appropriate film-forming material from a volatile solvent-based solution followed by evaporation of the solvent. A water or water-ethanol solution is optimal because it does not attack and deform the EVA surface. The usual organic solvent solutions of a polymer are unsuitable because the solvent interacts with the EVA and spoils its surface.
Three materials have been found suitable for this application, although other candidates for water or ethanol solution film formers are possible, including variations of these materials. The EVA used is DuPont Elvax 410. The film materials used are polyvinyl alcohol (Mowiol 40-88 Hoechst, mw 127,000, Aldrich Chemical Co. 32,459-0, CAS 9002-29-5), polyvinylpyrrolidone (PVP, Fisher Scientific Co. BP431-100, mw 40,000, CAS 9003-39-8), and chitosan (Aldrich Chemical Co. 44,887-7, medium mw, CAS 9012-76-4). These are used in solution in water or 50% ethanol in water at concentrations from 0.1 g/100 ml to 10 g/100 ml, most commonly 1 g/100 ml. The chitosan solutions contain 5% by volume acetic acid in the water to solubilize the chitosan.
To apply the coatings evenly it may be necessary to prepare the surface of the EVA by plasma etching to make it solution wettable. Also, some coating materials, particularly PVP, require etching of EVA to give satisfactory film adhesion. Exposure for about one minute to a plasma in air at a pressure of approximately one-half torr is sufficient. The articles to be treated are placed in a six-inch diameter glass vacuum desiccator with internal electrodes, connected to a vacuum leak-testing probe as a high-voltage high-frequency current source.
The coating solution can be applied by spraying, dipping, or adding exact volumes to a surface with a micropipet. Applying a measured volume of solution of known concentration over known surface area, as on caps, can control the film thickness added to the known area.
It has also been proposed to accomplish laser capture microdissection utilizing a conical surface. In this application, successive application and rotation of a conical surface enables a high concentration of similar cells at closely spaced intervals over the flat surface. Utilizing this disclosure, rod shapes can be coated completely.
It has also been proposed to provide such rods with spacer bands. These spacer bands contact the specimen or slide surface and hold the low temperature melt film a given distance away from the specimen. Such spacer bands can be protected against non-specific transfer with the coatings set forth herein. Dipping, with rotation to improve evenness of coating application may be used to evenly coat the spacer bands. Rods can also be coated by addition of solution from a micropipet during rotation.
Pattern coating, or control of the area coated by a film can be done by masking during the plasma etching process so that the non etched surface of EVA is not wetted by the solution. Various methods of printing transfer of solution to the surface can also be used to apply the solutions to coat the EVA in selected regions. Addition of ethanol to the solution of coating material will vary the wettability of the solution on the EVA.
The thickness of the film can be estimated by calculation from the volume and concentration added to a known area. The thickness of the film can also be measured microscopically by counting interference fringes with the film against a glass slide surface or by measuring the focus location change when focusing on the upper and lower surface of a film. Inclusion of a dye, preferably fluorescent, in the film can be used to calibrate the thickness by color intensity measurement. A dye also makes it easy to see the location of the film, which would otherwise be difficult. An example dye is rhodamine B (CAS 81-88-9) in a concentration of 100 micrograms/ml in the solution.